Synthesis of Small Particles

ABSTRACT

The invention provides an apparatus for forming fine particles of a substance in a precipitation chamber, in which the apparatus has means to convey the fine particles from the precipitation chamber to at least one particle collection chamber, downstream of the precipitation chamber, the particle collection chamber having an inlet and an outlet separate from the inlet. The invention also provides a method of forming fine particles of a substance, the method comprising contacting a non-gaseous fluid containing the substance with a dense fluid to expand the non-gaseous fluid in a precipitation chamber, conveying a resulting mixture of fluid and the fine particles from the precipitation chamber to a collection chamber, the collection chamber having an inlet and an outlet separate from the inlet.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/497,715, now issued as U.S. Pat. No. 7,641,823; which is a 35 USC§371 National Stage application of International Application No.PCT/AU02/01657 filed Dec. 6, 2002; which claims the benefit under 35 USC§119(a) to Australia Patent Application No. PR9382 filed Dec. 7, 2001.The disclosure of each of the prior applications is considered part ofand is incorporated by reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for forming and collectingfine particles of a substance, such as a pharmaceutical or biologicalsubstance, by anti-solvent precipitation, particularly but notexclusively suitable for administration to organisms. The invention alsorelates to fine particles of biological substances produced by themethod and to compositions, particularly pharmaceutical compositions,containing an active substance.

2. Background Information

Throughout this specification, unless stated otherwise, where adocument, act or item of knowledge is referred to or discussed, thisreference or discussion is not an admission that the document, act oritem of knowledge, or any combination thereof, at the priority date, waspart of the common general knowledge.

Production of uniform micron size particles (or within a narrow sizerange) of fragile molecules such as proteins is a challenge in thepharmaceutical industry. One use of fine particles is pulmonaryabsorption of drugs. This is an important route of entry for manyindications including some pulmonary diseases, for example, bronchialasthma. One advantage of this mode of administration is that access tothe circulation is rapid, because the surface area is large. As well asalmost instantaneous absorption of the drug into the blood, delivery tothe lung has the advantages of avoidance of hepatic first-pass loss, andin the case of pulmonary disease, local application at the desired siteof action.

Delivery to the lung may also provide an alternative for the treatmentof conditions that have traditionally been treated by systemicadministration of a drug. The administration of proteins is a case inpoint. Insulin is currently administered by injection because it is notstable in the gastrointestinal tract. Diabetic patients need toself-administer several injections. However, there is a lack ofcompliance with the use of injections because of the associatedinconvenience and pain. Administration of the protein to the lung ismore likely to be accepted by such patients and is therefore anattractive alternative to injections, as long as the protein can beformed as fine particles, without significant loss of biologicalactivity. Usual criteria for the use of aerosol delivery for theadministration of therapeutic drugs to the lung are that the drug is inparticulate form with the particles having a size in the range of about0.05-10 μm, preferably 1-5 μm while (obviously) retaining biologicalactivity, which often requires the substance's structure to bemaintained. A common problem in manufacture of such particles isunacceptable variation in particle size.

Drugs in the form of fine particles are also suitable for use in thearea of oral, controlled or sustained release delivery. One applicationof such technology is in the case of a drug in which there is a smalldifference in dosage levels between the drug being effective and beingtoxic. In the latter technology, it is also important that the particleshave a uniform particle size.

Another application of fine particles of pharmaceuticals is transdermaldrug delivery. Apart from traditional sub-cutaneous, intravenous, etc.injection, new methods of administration are being used, such as lasersto create a fine channel through the skin for drug delivery. A similarmechanism involving high-pressure drug delivery transdermally is alsobeing used. Thus, the applications for fine or micron-sizedpharmaceutical particles are increasing.

Dense gas techniques utilizing fluids, near or above their criticalpoint, as a solvent or anti-solvent have been developed in recent years.Two dense gas methods have been considered for the production of solidparticles. The first method is known as the Rapid Expansion ofSupercritical Solutions (RESS), and involves expanding a supercriticalsolution of the material of interest through a nozzle. Whilst providingan effective method for producing some fine particles, the applicabilityof the RESS method is limited by the low solubility of proteins in densecarbon dioxide.

The second method, known as the gas anti-solvent process, involvesrapidly precipitating solutes from organic solutions, typically usingdense carbon dioxide as an anti-solvent. The anti-solvent expands thesolution, thereby decreasing the solvation power of the solvent, andeventually resulting in the precipitation of the solute.

Gas anti-solvent processes have been utilized for the generation ofmicron-sized particles in two modes. The first mode, known simply as thegas anti-solvent process (GAS), involves the gradual addition of ananti-solvent to the organic solution containing the solute until theprecipitation occurs. The second mode, known as the Aerosol SolventExtraction System (ASES), involves continuous introduction of a solutioncontaining the solute of interest through a nozzle into a flowing densegas stream. As the solution is sprayed in to the dense gas, high degreesof supersaturation result in the precipitation of fine particles. Ingeneral, precipitation using this process is rapid and requires mildoperating temperatures and pressures.

The GAS process has been attempted for the generation of micron-sizedparticles of insulin, lysozyme, and peroxidase. The difficulty ofapplying these techniques to the production of micronised particles ofpH sensitive proteins is that they involve exposure of the protein toorganic solvents, the latter being potential denaturants. This would,for example, inactivate insulin. Organic solvents are also undesirableas they are more difficult to dispose of. Thus, this process is largelyunsuitable.

In one attempt to overcome this limitation, a form of the ASES processhas been developed, referred to as Solution Enhanced Dispersion bySupercritical Fluid (SEDS). SEDS involves using the ASES process butwith a special coaxial nozzle which, in part, overcomes the problem ofexposure to organic solvents.

Current apparatus utilising these processes, particularly ASES, for theproduction and collection of particulate products comprise aprecipitator and a collection device in the same chamber. The solutioncontaining the product of interest and the anti-solvent (which containsthe dense gas and, optionally, a modifier) are passed through theprecipitation chamber co-currently. As the particles are formed, theyfall to the bottom of the collection device under gravity and can becomecompacted, aggregated (physical association) or agglomerated (chemicallybonded). The particles can also become further compacted during thewashing stage at the end of the process, due, for example, to the highpressure and high flow-rate of the dense gas anti-solvent.

Aggregation occurs when a collection of two or more particles are heldtogether by weak cohesive forces, such as van der Waal's forces.Aggregates can be dispersed with shear forces and/or solvents.Agglomeration on the other hand, occurs when a collection of two or moreparticles are held together by strong inter-particle forces such ascrystal bonds. Agglomerates are more difficult to break up and disperse.

In small particle formation processes, it is desirable to avoid theparticles becoming agglomerated or compacted, since it is more difficultto break this material up, particularly while avoiding damage to theactive component. The particles resulting from such processes are,therefore, not uniform in size and shape, which is not ideal for the useof such particles in pharmaceutical applications. However, some degreeof aggregation may be desirable in some situations where the particlesproduced are too fine to be collected. The fine powders that have notbecome aggregated may be washed out of the system, resulting in a lowyield. Aggregation between particles makes the particles larger andeasier to collect, and after collection the aggregate can be broken upby mechanical force.

Particles to be used for the pulmonary delivery of pharmaceuticalsshould ideally be less than 5 to 10 μm in diameter. Particles of thissize are more easy to aerosolise, and when inhaled, these particles areeasily able to reach the lungs. However, when particles become compactedin the collection chamber, the mass fraction of particles with adiameter of less than 5 μm (and thus suitable for pulmonary delivery) islow.

Collection processes using known single-stage apparatus are essentiallybatch processes with short run times, due to the necessity of regularlystopping the run to remove the precipitated particles before caking(i.e., aggregation of a mass of fine particles which may form a block toa chamber's outlet) occurs. The production of particles using suchapparatus is thus, necessarily, a batch-wise process. The process istherefore inefficient and there can be poor yields and recovery of theproduct.

The invention is directed towards an apparatus for particle formationwhich operates in a more efficient manner (i.e., increase the yield offine particles collected relative to starting materials) and does notdamage the particles that are formed using the apparatus orsubstantially increase the average particle size collected.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides an apparatus forforming fine particles of a substance in a precipitation chamber, inwhich the apparatus has means to convey the fine particles from theprecipitation chamber to at least one particle collection chamber,downstream of the precipitation chamber, the particle collection chamberhaving an inlet and an outlet separate from the inlet. In oneembodiment, the outlet is disposed above the inlet in use of theapparatus, such that gravity exerts a force generally towards the inleton particles adjacent the outlet.

The particles are formed by contacting a non-gaseous fluid containingthe substance with a dense fluid to expand the non-gaseous fluid in theprecipitation chamber.

In a further form of the invention, the apparatus further includes atleast two particle collection chambers in parallel with each other andeach able to be connected in series with the precipitation chamber.

The invention also provides a method of forming fine particles of asubstance, the method comprising contacting a non-gaseous fluidcontaining the substance with a dense fluid to expand the non-gaseousfluid in a precipitation chamber, conveying a resulting mixture of fluidand the fine particles from the precipitation chamber to a collectionchamber, the collection chamber having an inlet and an outlet separatefrom the inlet.

In one embodiment, the method is conducted wherein the fine particlesflow with the dense gas from a first chamber in which the particles areformed to a second collection chamber, from which the particles arecollected. Preferably, the second collection chamber has an inlet and anoutlet separate from the inlet, in which the fine particles and densefluid pass through the inlet and the flow of dense fluid through theoutlet is adjusted to maximise the proportion of fine particlescollectable from the second collection chamber.

In an aspect of the invention, there is provided an apparatus whereinthe precipitator and the collector are two separate chambers, in whichfine particles (with a narrow particle size distribution) can beproduced which are less affected by the problems of compaction,agglomeration and aggregation, while still being collectible in anadequate yield. In addition, where the particles are finer thanparticles produced using prior art apparatus, and the mass fraction ofparticles with a diameter of <5 μm is higher than in prior artapparatus, an adequate yield is obtainable. Also, an essentially batchprocess can be made semi-continuous.

In another aspect, the invention provides an apparatus for the methodsdescribed above. In particular, there is provided an apparatus forforming fine particles including:

-   -   a precipitation chamber in which the dense fluid and non-gaseous        fluid containing the substance are contacted so as to        precipitate the fine particles; and    -   at least one particle collection chamber, downstream of the        precipitation chamber from which the fine particles are        collected.

Preferably, the particle collection chamber has an inlet and an outletseparate from the inlet, in which the fine particles and dense fluidpass through the inlet and the flow of dense fluid through the outlet isadjusted to maximise the proportion of fine particles collectable fromthe second collection chamber.

It is also desirable for the apparatus to include at least two particlecollection chambers in parallel to be used alternately, each in serieswith continuous use of the precipitation chamber in afill-empty-fill-empty cycle.

In one embodiment, within the collector device or collection chamber,the particles are largely suspended by the force exerted on them by aflow of dense gas in one direction, which force is generally balanced bya second force. This second force may be gravity (i.e., the particles'weight) where the collection chamber is orientated such that the outletis above the inlet when the collection chamber is connected to (oron-line with) the particle formation apparatus. Such a force could alsobe generated by other means, e.g., centrifugal force with an appropriatearrangement of the collection chamber(s), and such an arrangement wouldallow variation of this second balancing force. Where the particles arecharged, electromagnetic forces may be employed. As will be appreciatedby one skilled in the art, the objective is to balance the force onparticles of the carrying fluid, which otherwise tends the particleseither to “cake” at one end of the collection chamber and/or to escapethrough the outlet with the carrying fluid.

Therefore, the newly formed particles do not “fall” on top of thepreviously formed particles and are not subjected to a pressure whichcould deform their shape and are also less susceptible to aggregation.

Further, the use of such an apparatus allows higher yield and recoveryof particles per run, the ability to process more material per run withlonger run times, all of which lead to a more efficient process andgreater production capacity.

Such an apparatus can be readily scaled up to process larger amounts ofmaterial.

The anti-solvent used in the method of the invention should be a neutralsolvent and/or a solvent of relatively low polarity. Suitable solventsinclude a C₁₋₄ alkane gas, a C₂₋₄ alkene gas, a C₂₋₄ alkyne gas,hydrofluorocarbons, refrigerants, like RF134a, and some organicsolvents, such as hexane, or two or more thereof. In one embodiment, theanti-solvent is an alkane gas. Ethane is a particularly preferredanti-solvent. Preferably, the anti-solvent does not significantly alterthe pH of the non-gaseous fluid.

The method of the present invention is capable of producing fineparticles of the substance, and is particularly useful for theproduction of fine particles of pH sensitive substances and biologicallyactive substances, since the biological activity of such substances maybe retained. The present method is also particularly suited to watersoluble substances. The non-gaseous fluid is an aqueous solution in oneembodiment.

The modifying agent may be present in an amount sufficient to absorbsubstantially all of the non-gaseous fluid of the non-gaseousfluid-biologically active substance solution. The modifying agent may beany substance that modifies the polarity of the anti-solvent and acts asan extractant for (i.e., solvent for) the non-gaseous fluid. Themodifying agent may be selected from the group consisting of C₁₋₆alkanols, C₁₋₆ thiols and C₁₋₆ amines. Preferably, the modifying agentis ethanol.

In one embodiment of the invention, the anti-solvent/modifying agentcombination is ethane/ethanol.

In another aspect, the invention provides smaller particles than arepossible from the prior art by use of a neutral anti-solvent modified bya modifier to change its polarity by using a separate collection chamberin a “dual stage” process, particularly where most of the newly formedfine particles are suspended within the chamber by the force of thedense fluid flowing through the chamber accommodating the particlesbeing balanced by gravity in the opposite direction, to reduceaggregation and agglomeration of the particles.

The dense gas can be at various temperatures and pressures. Preferablythe temperature of the dense gas is in the range of −20° C. to about100° C., most preferably about 5° C. to about 45° C. The lowertemperatures result in increased viscosity an reduced mass transferproperties, and this reduces efficiency of the method. High temperaturesare more costly to run and may damage the substance. Preferably thedense gas has a pressure in the range of about 1 bar to 400 bar. Apressure between about 5 to 200 bar is particularly preferred. Mostpreferably, the pressure of the dense gas is such as to maintain themixture of solvent, anti-solvent and modifying agent as a single phasewhich reduces loss of precipitate which may remain dissolved in a secondphase, and be washed from the system.

Preferably, both the anti-solvent gas and the modifier are substantiallyinert to the pH-sensitive, biologically active substance.

The particles produced by the method of the invention may also includedelivery agents such as liposomes, lipids (including phospholipids),water soluble polymers, controlled-delivery coatings, surfactants,phytosterols, and any other delivery agents known in the art.

Preferably, at least half, and more preferably substantially all, of thefine particles produced by the method of the invention have a particlesize less than 10,000 nm. More preferably, the fine particles have asize no greater than 6,500 nm. Particles having a size in the range ofup to 5,000 nm are particularly useful for administration to the lung.If smaller particles are desired, it is believed that the method of thepresent invention can produce particles down to nanometre size, althoughsuch particles can be more difficult to collect and naturally aggregateinto larger particles.

The solution of the active substance may be contacted with dense gas inany suitable manner. Preferably, the solution is introduced as dropletsinto the dense gas. For example, the solution and dense gas may becontacted by concurrently spraying the two through a nozzle or the like.Alternatively, the solution may be sprayed through the dense gas. Afurther option is to pass the solution concurrently or countercurrentlywith respect to a stream of the dense gas. The solution may be passedthrough a continuum of the dense gas in the form of a thin film orplurality of streams.

Preferably the method of the invention is carried out using the ASESprocess. The term “pH-sensitive, biologically active substance”, as usedthroughout the specification, refers to any natural or syntheticsubstance which possesses a biological activity such as, for example, anenzymatic activity, channel function (e.g. ion channel), receptor orbinding function, hormonal or neurotransmitter activity, or otherpharmacological activity, or a protein, polypeptide, peptide, peptideanalog or peptidomimetic, or nucleic acid or nucleic acid in associationwith a protein, polypeptide or peptide, which is adversely affected bypH outside of the normal physiological pH range (e.g. 6.8 to 7.5),especially low pH (e.g. less than 5.0). The adverse affect upon thebiological activity caused by the pH may be the result of, for example,degradation, cleavage or conformational changes in the substance orinactivation of an active site or binding domain.

The pH-sensitive, biologically active substance is preferably selectedfrom the group consisting of an antimicrobial agent, virus, antiviralagent, antifungal pharmaceutical, antibiotic, nucleotide, DNA, antisenseDNA, RNA, antisense RNA, amino acid, peptide, protein, enzyme, hormones,immune suppressant, protease inhibitors, thrombolytic anticoagulant,central nervous system stimulant, decongestant, diuretic vasodilator,antipsychotic, neurotransmitter, sedative, anaesthetic, surfactant,analgesic, anticancer agent, anti-inflammatory, antioxidant,antihistamine, vitamin, mineral, sterol, phytosterol, lipid and estersof fatty acids.

More preferably, the pH-sensitive, biologically active substance isselected from proteins, polypeptides, peptides, peptide analogs orpeptide mimetics. Most preferably, the pH-sensitive, biologically activesubstance is selected from the proteins insulin, erythropoetin,calcitonin, LHRH, somatostain, epidermal growth factors, DNase plateletderived growth factors, interleukins, interferons, cytokines, peptidesof immunoglobulins, TNF and other biologically active peptides,monoclonal antibodies based on TNF inhibitors as well as antibodiesbased on inhibitors of cytokines and interleukins.

In a second aspect, the present invention provides a pharmaceuticalcomposition comprising particles of a biologically active substanceproduced by the method of the present invention.

The pharmaceutical composition is preferably in a form suitable forinhalation delivery, for example, for delivery by a metered dose inhaleror a nebuliser. Further, a transdermal delivery system may be used(e.g., recent devices involving laser-generated or high-pressure dermalchannels) and more traditional parenteral administration.

In a third aspect, the present invention provides a method of treatmentof a subject, the method comprising administering to the subject, aneffective amount of particles of a biologically active substanceproduced by the method of the present invention.

The method of the third aspect may be the treatment of insulin-dependentdiabetes by administration of insulin particles produced by the methodof the present invention.

In the description, the term “dense gas” means a fluid substantiallynear or above its critical pressure (Pc) and temperature (Tc). Inpractice, the pressure of the fluid is likely to be in the range(0.5-1.5)Pc and its temperature (0.5-1.2)Tc.

It will be understood that the term “comprises” (or its grammaticalvariants) as used in this specification is equivalent to the term“includes” and should not be taken as excluding the presence of otherelements or features.

The method of the present invention, in its preferred forms, may provideone or more of the following advantages:

-   -   1. The ability to produce and collect significant yields of fine        powders of proteins and other pharmaceuticals with narrow        particle size distributions.    -   2. The ability to use aqueous solutions thereby enabling        concentrated solutions of material to be processed with minimal        risk of deactivation of biological activity. Aqueous solutions        are also easier and cheaper to handle.    -   3. The use of one of the preferred anti-solvents, ethane,        overcomes the problems associated with an acidic pH environment        for proteins and other pH sensitive or acid-labile molecules        (ethane is neutral).    -   4. The use of an organic compound such as ethanol as the        modifier in the ethane phase appears to enhance the        morphological characteristics of the powders produced, including        insulin. While not wishing to be bound by any particular theory,        it appears that the morphological characteristics of the powders        produced are also dependent on the relative concentrations of        the solutions at the time of contact, the time period that the        solutions are in contact with each other, and the time period        that the particles are in contact with each other after        precipitation. These variables can be adjusted during use of the        method to optimise results.    -   5. The ability to use a substance such as ethanol as a modifier        for the anti-solvent as described in 3 above, yet produce fine        particles of a biologically active substance in which 98 to 100%        biological activity has been retained.    -   6. The ability to introduce an additional component in either        the solvent stream or the modified anti-solvent stream, which        when co-precipitated with the protein or pharmaceutical will        enhance dissolution rates and/or bioavailability.    -   7. The ability to process materials at temperatures below those        required for supercriticality, thereby reducing the risk of        thermal degradation.    -   8. The ability to work at lower pressures than that claimed in        the prior art, thereby reducing the potential cost of the        process.

In order that the invention may be more readily understood, we providethe following non-limiting embodiments as examples.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a laboratory scale embodiment of the invention,having a separate particle collection chamber, called a “dual stage”apparatus.

FIG. 2 shows a graph comparing the aerodynamic particle sizedistribution of insulin particles precipitated at 25° C. and 150 barusing 20 mol % ethanol in carbon dioxide and 30 mol % ethanol in ethane.

FIG. 3 shows an HPLC chromatogram of the insulin monomer.

FIG. 4 shows an HPLC chromatogram for the separation of insulin anddeamido insulin.

FIG. 5 shows an SEM of insulin particles.

FIG. 6 shows a graph which represents the aerodynamic particle sizedistribution of insulin powder.

FIG. 7 illustrates an industrial scale embodiment of the inventionsimilar to that in FIG. 1, having a separate particle collectionchamber, also called a “dual stage” apparatus.

FIG. 8 describes another embodiment of the invention, being an apparatushaving more than one particle collection chamber.

While suitable for any of the proteins mentioned above, the examplesillustrating the invention are described using insulin as the desiredactive ingredient. Similarly, for the purposes of illustration, theexamples describe the use of ethane as the anti-solvent and ethanol asthe modifier. Low endotoxin bovine insulin (lyophilized powder of 28.5USP units/mg, lot No. 47H0573) and sodium hydroxide were purchased fromSigma Chemicals and used as received and dissolved in deionised water.Liquid carbon dioxide and ethane (Industrial Grade 99.95%) werepurchased from BOC Gases.

The set-up for the ASES apparatus (FIGS. 1, 7 and 8) was designed toimprove the proportion of fine particles generated of the drug, whichcould be collected as such, and increase the yield and recovery of theproduct. In order to minimize compaction and increase the fine particlemass (FPM) fraction of the precipitate, a dual precipitation andcollection chamber arrangement with no filter between the precipitationand collection chambers was used. The precipitates and anti-solvent passco-currently from the precipitation chamber to the (second ordownstream) collection chamber. The embodiment in FIG. 8 has two“parallel” collection chambers to enable continuous (rather than batch)operation by alternating between the two collection chambers.

An ASES apparatus used is schematically shown in FIG. 1. The apparatusincludes a precipitation chamber 1 which is fitted with a nozzle 2.Through this nozzle is sprayed a solution of the substance of interestand the dense gas and the modifier. The solution of the substance ofinterest is pumped into the precipitation chamber from the reservoir 3by means of pump 4. The dense gas is pumped into a static mixer 5 bymeans of pump 6. The modifier is simultaneously pumped from itsreservoir 7 into the static mixer 5 by means of pump 8. The desiredmixture of ethanol with ethane is prepared in the static mixer 5. Thechamber is first pressurised with carbon dioxide via a syringe pump(ISCO Model 500) 6 to attain a pressure of 20 to 180 bar to maintain theethane/ethanol mixture as a single phase. The modified ethane is thendelivered into the precipitation chamber 1 at the desired processingconditions and CO₂ is purged from the system. The operating temperatureis controlled to within ±0.1° C. using a temperature controlled waterbath heated by heater 12.

On leaving the static mixer, the dense gas/modifier mixture is passedthrough a cooling coil 9, and then into the precipitation chamber 1. Theflow of the fluids continues through the precipitation chamber and to asecond high-pressure chamber, the particle collection chamber 14. Theflow rate is controlled by the metering valve 11. The apparatus isplaced in a water bath, which is heated by the heater 12, to control andmaintain the temperature of the precipitation and collection chambers.There is no filter between the two chambers, but a filter is downstreamof the particle collection chamber.

The flow of the fluids through the precipitation chamber continues intothe collection chamber 14 via its inlet (at the bottom of collectionchamber 14 as shown in FIG. 1). The dense fluid then passes through thecollection chamber outlet to filter 10. The flow rate is controlled bythe metering valve 11. Once passing through the valve, the flow offluids passes through a cold trap or separator 13, at low temperature,to separate the solvent and modifier from the dense gas. The dense gascan then be recycled through the system.

Once the desired temperature and pressure (namely 25° C. and 150 bar)are achieved in the chamber 1, and the chamber filled with 30 mol %ethanol in ethane mixture, the aqueous solution containing the protein(low endotoxin bovine insulin (lyophilized powder of 28.5 USP units/mg,lot No. 47H0573, purchased from Sigma) is pumped from reservoir 3 at aconstant flow rate using a solvent delivery unit (Waters pump, Model510) 4 and sprayed through a capillary nozzle 2 (50 um internaldiameter) into the chamber. The pressure drop through the nozzle wasadjusted to about 50 bar by a metering valve 21. This pressure drop canbe adjusted to optimise the efficiency of the process. Modified ethanewas fed continuously through to the chamber at a constant flow rate thatwas adjusted with the metering valve 11. The operating conditions, theflow-rate ratio of the aqueous feed and the anti-solvent, and themodifier mole fraction were optimized (using published ternary phaseequilibrium data for ethane, ethanol and CO₂) so as to have a homogenous(i.e., single phase) mixture of dense fluid-ethanol-water in theprecipitation chamber.

The mole fraction of ethanol in the anti-solvent was kept at 0.3 and avolumetric flow rate ratio of feed to anti-solvent of 0.4/12 was used inthe process. This is primarily adjusted so as to maintain a singlehomogeneous phase in the system, particularly in the precipitationchamber. The high flow rate of the anti-solvent facilitated thedispersion and mixing of the aqueous spray mist across the chamberresulting in higher rates of water extraction from the droplets.

Micronised particles of proteins with uniform particle size suitable foraerosol drug delivery systems were thus produced from aqueous solutionat room temperature in effectively one step. No toxic chemicals wereused. Residual ethanol content in the final product was less than 10ppm. The small particulate material of the present invention isparticularly useful in the preparation of pharmaceutical preparationsformulated to provide oral, controlled or sustained release, or forinhalation or transdermal administration and conventional modes.

FIG. 8 shows a modified design incorporating two particle collectionchambers 14. The apparatus operates as described for FIGS. 1 and 7, butonce the first particle collection chamber has been filled to optimumcapacity for collection of particles, the flow of dense gas containingthe formed particles is diverted into a second particle collectionchamber. The removal of the particles from the first particle collectionchamber can be effected while the apparatus, particularly theprecipitation chamber, is still in operation. Once the second particlecollection chamber has been filled to capacity, the flow of dense gascontaining the precipitated particles can be diverted into a thirdparticle collection chamber, or back to the first particle collectionchamber which by this time would have been cleaned out. In this way, theapparatus can operate in a continuous manner for the production ofparticles.

On the laboratory scale, the view cell (such as a Jerguson sight gauge,model 13-R-32) can be used as a precipitation chamber for visualobservation of the precipitation stages as shown in FIG. 1. A coaxialnozzle 2 is connected to the chamber for spraying the solutions andanti-solvent. The nozzle consists of a capillary tube (SGE, PEEK tube200 mm length, 50 μm i.d., and 1.59 mm o.d.) inserted into a stainlesssteel tube (Alltech, 2.16 mm i.d. and 3.18 mm o.d.). The three pumps inthe system are for the delivery of the protein solution (Waters Model510), ethanol (Hewlett Packard, series 1050) and anti-solvent such asethane and CO₂ (ISCO Syringe pump 500D). The anti-solvent was mixed withethanol in line using a static mixer (KOFLO Corporation). Thecomposition of the mixture was adjusted by the flow rate of each pump.The anti-solvent flow rate was controlled by a metering valve at theexit. The ethanol/water/anti-solvent mixture was maintained in ahomogeneous phase at the operating pressure and temperature of theprocess. The carbon dioxide/ethanol mixture was passed through apreheating coil to attain the system temperature. The high pressurechambers were placed in a water bath consist of a thermostatic heater(Thermoline Unistat heater/circulator) to control the temperature. Thepressure of the system was monitored with Druck pressure transducers(Model PDCR 911) coupled to Druck pressure indicators. The anti-solventand solvent were separated after the metering valve and the solvent wascollected in a chamber. The filters were placed after the high pressurechambers to collect any remaining fine powder in the line.

Preparation of Particles

The preparation of the particles was carried out using the apparatusrepresented in FIG. 1 (discussed above). The desired mixture of ethanolwith ethane is prepared in the static mixer 5. The chamber is firstpressurised with carbon dioxide (which aids apparatus sterilisation) viaa syringe pump (ISCO Model 500) 6 to attain a pressure of 20 to 180 barto maintain the ethane/ethanol mixture as a single phase. The modifiedethane is then delivered into the precipitation chamber 1 at a pressureof 140 bar, a temperature of 35° C., and a flow rate of 15 mL/min, andCO₂ is purged from the system. The operating temperature (35° C.) iscontrolled to within ±0.1° C. using a temperature controlled water bathheated by heater 12.

Micronisation by the ASES process was conducted by first placing thehigh pressure chambers in the water bath and adjusting the temperatureof the system. After the system approached the temperature of theprocess, the pressure of the system was adjusted by adding anti-solventto the chambers from the top. The anti-solvent flow rate was thenadjusted by the metering valve at the exit. The required amount ofethanol was then added to the system by controlling the flow rate ofeach line. When using CO₂ as the anti-solvent, to achieve a CO₂-20 mol %ethanol mixture (ie, the CO₂:Ethanol molar ratio was 1:4), a flow rateof 3.4 mL/min and 15 mL/min of ethanol and CO₂, respectively, werepassed through the static mixer. When using ethane as the anti-solvent,an ethane-30 mol % ethanol mixture was prepared using flow rates of 15mL/min and 2.4 mL/min for ethane and ethanol, respectively.

After the system approached steady state, 0.1 mL/min insulin solution(100 mg/mL) was sprayed through the inner nozzle. The solution disperseddue to the high flow rate of the anti-solvent. Extraction of the waterwas facilitated from the droplets by the ethanol and fine insulinparticles were formed. The metering valve 11 at the exit (ie, justdownstream of the collection chamber outlet) is adjusted onceprecipitation commences such that the force exerted on the particles inthe particle collection chamber by the dense gas flowing upwardlythrough the collection chamber is balanced by their weight (by gravity)so that the particles are in effect suspended within the collectionchamber and not compacted. This effect is achieved with a flow rate of15-20 mL/min. Other similar arrangements may be contemplated, such ascollection chambers rotating about an axis to generate a force counterto that of the dense gas flowing through the collection chamber. Upondisconnection from the precipitation chamber, the collection chamber isdepressurized and the product collected from both chambers, sealed inairtight containers and stored in the freezer (−18° C.).

When the entire apparatus is shut down, it has been found desirablethat, after spraying, the solution ethanol and water residues wereremoved from the precipitate by passing the equivalent of 5 chambervolumes of ethane at operating pressure and temperature through thecollection chambers. A small amount of ethane (about 10-50 mL) was alsopurged through the nozzle to remove any remaining solution. This processis necessary prior to the depressurisation of the system to prevent anydroplets of the aqueous solution retained in the nozzle falling into theprecipitation chamber, contacting the precipitated particles and causingagglomeration of the particles.

The effect of apparatus design on the characteristics of insulinparticles precipitated from aqueous solution using ethane/30 mol %ethanol was examined at 25° C. and 155 bar. This is discussed furtherbelow.

It has been demonstrated that processing of insulin using ethane-ethanolprovides for the retention of biological activity as indicated by the invitro test for monomer content. Insulin processed with CO₂-ethanolexperienced significant deactivation in this respect. In a single stage(prior art) apparatus, the particle characteristics of the CO₂-ethanoland ethane-ethanol processed material are similar as shown in Table 1.In the dual stage process (illustrated above and FIGS. 1, 7 and 8) thefine particle mass for the CO₂-ethanol system is similar to thatobtained for the single stage process. A dramatic increase in thisparameter was obtained for material processed with ethane-ethanol.

TABLE 1 Data from Single Stage and Dual Stage Production Units SingleStage Dual Stage Anti-Solvent D_((0.5, V)) FPM(%) D_((0.5, V)) FPM(%)Ethane-ethanol 12.8 +/− 1.0 20.0 4.8 +/− 0.4 43.5 CO₂ - ethanol 11.8 +/−1.6 20.3 8.2 +/− 0.6 23.1 D_((0.5, V)) Median particle size based onvolume (i.e. below which 50% of particles occur. FPM(%) Fine ParticleMass - mass fraction of particles below 5 μm according to CascadeImpactor tests.

Whilst some reduction in D_((0.5,V)) occurs with the 2-stage CO₂-ethanolprocessing method, only 21.9% of this material can be dispersed toproduce material less than 5 μm aerodynamic diameter. The materialobtained using the 2-stage ethane-ethanol process has a dramaticallyreduced D_((0.5,V)), consistent with the 100% increase in the amount ofmaterial less than 5 μm aerodynamic diameter, as indicated in Table 1.

By way of another comparison, insulin was precipitated in a single-stageapparatus as nano-sized particles, most of which ranged in size from50-500 nm using the apparatus represented in FIG. 1. Particle sizedistribution studies showed that the particles agglomerated to formmicron-sized particles. The fine particle mass of the micronised powderprecipitated was only 20%. The particles were collected on the filterassembly at the bottom of the chamber. Due in part to the high pressureand high flow rate of the anti-solvent, the powder was compacted anddifficult to disperse. The fine particles that were not aggregatedwashed from the system by passing through the filter, therefore theyield was low (40%).

Insulin was also precipitated as nano-sized particles ranged in sizefrom 50-500 nm using the apparatus represented in FIG. 1. The fineparticle mass of insulin processed by the modified ASES apparatus wasincreased to 45%. The filter at the bottom of the first chamber waseliminated, producing a precipitate which was less compacted. Theprecipitate moves downward from the precipitation chamber and is carriedupwardly part way through the collection chamber where gravity acts onit counter to the anti-solvent (dense fluid) flow direction, suspendingthe particles in the chamber, which minimises their compaction. Additionof the second (i.e., particle collection) chamber enabled improvedrecovery of fine particle mass and increased the yield to 90%. FIG. 2shows the aerodynamic particle size distribution of insulin powders inthe single chamber and the dual chamber apparatus.

In order to determine whether the biological activity of the insulin wasretained after the particle formation process, the biochemical integrityof the insulin powder was assessed using size exclusion chromatography.A protein-Pak 125 column (Waters, USA) was used for insulin. The mobilephase consisted of 50 mM sodium phosphate buffer at pH 3 with 300 mMsodium chloride.

The powder samples were dissolved to prepare 1 mg/mL solution indeionised water by gentle shaking for 10 minutes to have completedissolution. The supernatant was filtered through 0.45 mm membranefilter and then injected into the HPLC column. The percent of monomerand the soluble aggregates were determined by comparing the peak area ofboth monomer and the high molecular weight soluble species in the samplewith the peak area of a standard concentration of 1 mg/mL. The percentof insoluble aggregates was estimated from the difference in the totalpeak area between the sample and the protein standard solution. Thesoftware Millennium 3.5 was used in the quantification of the monomercontent. The HPLC spectrum of insulin is shown in FIG. 3. The monomercontent of the sample was 99.5% thus providing an in vitro measurementillustrating that retention of biological activity was almost complete.

In order to determine the percentage of insulin that was deamidated bythe process, a test of the insulin was performed using high performanceliquid chromatography (HPLC). A reverse phase column (Symmetry® C₁₈, 5μm packing, 4.6 mm×150 mm) was used to separate the deamidated from thenon-deamidated insulin. The gradient method was used with eluents 0.1%trifluoroacetic acid/acetonitrile 26% to 33% and the flow rate of 1mL/min. The absorbance was monitored at 280 nm. The percentage ofdeamido insulin was determined by comparing the peak area of thedegradation product to the area of standard insulin containing the sameconcentration of insulin (2 mg/mL). The HPLC chromatogram for theseparation of insulin and deamido insulin is depicted in FIG. 4. Thepercentage of deamido insulin determined from the HPLC chromatogram was3.0, thus providing additional in vitro evidence for the retention ofbiological activity and chemical integrity of the molecule afterparticlisation by precipitation.

SEM images (FIG. 5) showed that insulin was precipitated as nano-sizedparticles ranging in size from 200 nm to 500 nm. These fine insulinparticles may aggregate to form larger particles due to intermolecularinteraction between the molecules. Analysing the particles by laserdiffraction studies showed that the insulin powder agglomerated andpossessed a median particle size of 5 μm (5000 nm).

In order to determine the aerosol performance of the collected fineprotein particles, in vitro testing of the particles was assessed usinga 5 stage Marple-Miller Impactor (Model 160, MSP Corporation, USA) withcut-off diameters of 10.0, 5.0, 2.5, 1.25, and 0.625 μm. TheMarple-Miller impactor consisted of a stainless steel inlet throat, fiveimpaction stages and an integral filter stage. The stages werepreviously coated with propylene glycol/methanol (50:50) to preventparticle bounce, thus minimising the probability of carry over of coarseparticles onto lower stages.

A mass of 10-20 mg of insulin was weighed into a gelatin capsule (size3, Park Davis, Australia) and was immediately dispersed by a powderinhaler device, Dinkihaler (Aventis, Frankfurt, Germany) into the MarpleMiller Impactor at 60 L/min using a vacuum source (ERWEKA GMBH,Germany). The fraction of the protein recovered in each stage wascalculated by measuring the absorbance of the protein solution againstthe absorbance of a standard 1% solution of insulin. The results of theassay method (measurement of relative UV absorption of a 1% solution)and the standard curves for insulin are indicated in Table 2.

TABLE 2 Protein Assay Methods and Absorptivity Value Determined from theStandard Curve Protein Assay method Standard curve Insulin UV absorbanceat 275 nm A_(1%) = 9.0 Conc. range = 0.01-0.1%

Insulin precipitated as nano-sized particles, was also agglomerated tomicron-sized particles. The median particle size (determined by laserdiffraction) was about 5 μm and the fine particle fraction (%<5 μm),determined by the Marple Miller Impactor, was at least 42%, as shown inFIG. 6. The fine particle mass (FPM) achieved is significantly greatercompared with the values reported in the literature. The maximum FPM forinsulin obtained by other methods was 10%.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

1-8. (canceled)
 9. A method of forming fine particles of a substance,the method comprising: contacting a non-gaseous fluid containing thesubstance with a dense fluid to expand the non-gaseous fluid in aprecipitation chamber to form the fine particles, conveying a resultingmixture of fluid and the fine particles from the precipitation chamberto a particle collection chamber by a carrying fluid, the particlecollection chamber having an inlet and an outlet separate from theinlet, wherein the carrying fluid, which comprises the dense fluid andthe non-gaseous fluid, exerts a first force on the fine particles toconvey the particles into the particle collection chamber, thecollection chamber being adapted such that a second balancing force isexerted generally towards the inlet on the fine particles adjacent theoutlet. 10-12. (canceled)
 13. The method according to claim 9 forforming fine particles of pH sensitive substances and biologicallyactive substances.
 14. The method according to claim 13 in which thedense fluid includes a modifying agent present in an amount sufficientto absorb substantially all of the non-gaseous fluid of the non-gaseousfluid-biologically active substance solution. 15-19. (canceled)
 20. Apharmaceutical composition comprising particles of a biologically activesubstance produced by the method according to claim
 9. 21. Apharmaceutical composition according to claim 20 in a form suitable forinhalation delivery, for example, by a metered dose inhaler or anebuliser.
 22. A method of treatment of a subject, the method comprisingadministering to the subject, an effective amount of particles of abiologically active substance produced by a method according to claim 9.23. A method of treatment of a subject, the method comprisingadministering to the subject, an effective amount of particles of abiologically active substance produced by a method according to claim14.
 24. A method for treating insulin-dependent diabetes byadministration of an effective amount of insulin particles produced by amethod according to claim 9.